Haploid embryogenesis

ABSTRACT

A switch to haploid embryogenesis is controlled by the activity of histone deacetylases (HDACs). Blocking HDAC activity with HDAC inhibitors (HDACi), e.g. trichostatin A (TSA), in  Brassica napus, B. rapa, B. oleracea, Arabidopsis thaliana  and  Capsicum annuum  male gametophytes leads to a large increase in the proportion of cells that undergo embryogenic growth. In  B. napus , treatment with one specific HDACi (SAHA) improves the conversion (i.e. germination) of these embryos into seedlings. Existing methods of culturing microspores of angiosperm plants following stress to produce haploid embryos, haploid plants and double haploid plants can be improved by adding HDACi to the culture medium. Advantageously, species hitherto recalcitrant to haploid embryogenesis via microspore culture are rendered useful when using HDACi. Haploid and double haploid plants are of industrial application in the plant breeding programmes.

This application claims the benefit of priority under 35 U.S.C. § 371 ofPCT International Application No. PCT/EP2014/070367, filed Sep. 24,2014, which in turn claims the benefit of priority to PCT InternationalApplication No. PCT/EP2013/069851, filed Sep. 24, 2013, which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The invention relates to the field of plant breeding and in particularthe generation and making of haploid or doubled haploid (DH) plants.More particularly the invention concerns the physico-chemical inductionof haploid embryos from plant gametophytes and the conversion of suchembryos into plantlets.

BACKGROUND ART

Many plant cells have the inherent ability to regenerate a completeorganism from single cells or tissues, a process referred to astotipotency. During sexual reproduction, cellular totipotency isrestricted to the zygote, which is formed in the seed from fusion of theegg and sperm cells upon fertilisation. Sustained division of the zygotegenerates the embryo, which contains the basic body plan of the adultplant. Establishment of groups of pluripotent stem cells in the stemcell niche of the embryonic shoot and root apical meristems ensures thecontinuous post-embryonic growth and development of new lateral organsthat is characteristic for plant development (see Bennett, T., andScheres, B. (2010) Curr. Top. Dev. Biol. 91: 67-102 and Besnard, F., etal. (2011) Cell. Mol. Life Sci, 68: 2885-2906).

Embryo development also occurs in the absence of egg cell fertilisationduring apomixis, a type of asexual seed development. Totipotency inapomictic plants is restricted to the gametophytic and sporophytic cellsthat normally contribute to the development of the seed and itsprecursors, including the unfertilised egg cell and surroundingsporophytic tissues (see Bicknell, R. A., and Koltunow, A. M. (2004)Plant Cell 16: S228-S245).

The totipotency of plant cells reaches its highest expression in tissueculture. The ability of a cell to undergo embryogenesis in vitro is bothan inherent and an acquired characteristic that requires the rightcombination of explant and culture environment. A wide variety of cellshave the potential to develop into embryos, including haploidgametophytic cells, such as the cells of pollen and embryo sacs (seeForster, B. P., et al. (2007) Trends Plant Sci, 12: 368-375 andSegui-Simarro, J. M. (2010) Bot. Rev. 76: 377-404), as well as somaticcells derived from all three tissue layers of the plant (Gaj, M. D.(2004) Plant Growth Regul, 43: 27-47 or Rose, R., et al. (2010)“Developmental biology of somatic embryogenesis” in: Plant DevelopmentalBiology-Biotechnological Perspectives, P. E-C and D. MR, eds (BerlinHeidelberg: Springer), pp. 3-26).

The treatments used to induce embryogenesis are diverse and range fromapplication of exogenous growth regulators to abiotic stress. Under theappropriate conditions; the explant resumes cell division and produceshistodifferentiated embryos, either directly from the explant orindirectly from callus. The morphological and cellular changes thatoccur during in vitro embryogenesis have been described in some species(Raghavan, V. (2004) Am. J. Bot. 91: 1743-1756; Seguí-Simarro, J. M.,and Nuez, F. (2008) Physiol. Plant. 134: 1-12), but there is still verylittle known about the initial steps involved in the acquisition andexpression of totipotency in individual cells, and many of the assumeddiagnostic features of cultured embryogenic cells are being revised inthe light of live imaging studies (Daghma, D., et al. (2012) J. Exp.Bot. 63: 6017-6021; Tang; X.; et al. (2013) J. Exp. Bot. 64: 215-228.

Molecular screens have been performed to identify the changes that occurduring in vitro embryogenesis, however the range of species, explantsand culture conditions that have been used, combined with low percentageof cells that form embryos, has made it difficult to develop a unifiedconcept of the totipotent plant cell.

In Arabidopsis, dynamic regulation of gene expression at the chromatinlevel has been shown to play a major role in translating thedevelopmental and environmental signals that regulate plant celltotipotency in planta (Zhang, H., & ©gas, J. (2009) Mol. Plant 2:610-627.

The basic structural and functional unit of chromatin is the nucleosome,which comprises DNA wrapped around a histone octamer, and associatedlinker histones. Nucleosomes can represent a physical barrier to DNA fornon-histone proteins due to the strong interaction between thepositively charged histones and negatively charged DNA. Transcriptionrequires physical binding of transcription factors to open DNA; thus,controlling the compaction and accessibility of loci through nucleosomesoffers a dynamic means to control gene expression. Dynamic changes inchromatin structure and gene transcription are mediated primarily by theinterwoven processes of chromatin remodelling and histone modification.Chromatin remodelling proteins use the energy from ATP hydrolysis toremove or reposition nucleosomes, while histone modifying enzymeschemically modify lysines and other amino acids on the exposedN-terminal tails of histones to change their charge and interaction withDNA and other proteins.

In plants, a number of conserved chromatin modifying proteins ensure thesuccessful transition from embryo development to post-embryonic growthby repressing pathways controlling embryo cell proliferation andidentity during germination. Loss-of-function mutants of these proteinsectopically express embryo identity genes and produce somatic embryosfrom seedlings. These chromatin modifying proteins include members ofthe Arabidopsis SWI/SNF and CHD class of chromatin-remodelling ATPases(Ogas, J., et al. (1999) Proc. Natl. Acad. Sci. USA 96: 13839-13844),members of the Polycomb Group (PcG) Repressive Complex 1 (PRC1) and 2(PRC2), which deposit repressive marks on histones, respectively,histone 2A lysine 119 (H2AK119) ubiquitination and histone 3 lysine 27(H3K27) trimethylation (see Chanvivattana, Y., et al. (2004) Development131: 5263-5276; Schubert, D., et al. (2005) Curr. Opin. Plant Biol, 8:553-561; Makarevich, G., et al. (2006) EMBO Rep. 7: 947-952; Chen, Z.,et al. (2009) Proc. Natl. Acad. Sci. USA 106: 7257-7262; Bratzel, F., etal, (2010) Curr. Biol. 20: 1853-1859; Bouyer, D., et al. (2011) PLoSGenet. 7: e1002014; Tang, X., et al. (2012) J. Exp. Bot. 63: 1391-1404).The large number of proteins that play a role in this process, combinedwith the potential cross-talk between different chromatin modifyingproteins (Zhang, H., et al. (2012) Plant Physiol. 159: 418-432) ensuresa multi-level dynamic control over cell totipotency.

Changes in chromatin organisation and modification are often associatedwith in vitro plant regeneration (Miguel, C., & Marum, L. (2011) J. Exp.Bot. 62: 3713-3725, but there are few examples where chromatin levelchanges are known to play a direct role in this process (He, C., et al.(2012) PLoS Genet. 8: e1002911).

Haploid embryogenesis was initially described almost 50 years ago inDatura stramonium (Guha, S., & Maheshwari, S. (1964) Nature 204: 497.The ability of haploid embryos to convert spontaneously or aftertreatment with chromosome doubling agents to doubled-haploid plants iswidely exploited as a means to generate homozygous plants in a singlegeneration, and has numerous breeding and trait discovery applications(Touraev, A., et al. (1997) Trends Plant Sci. 2: 297-302; Forster et al.(2007) supra). Haploid embryo production from cultured immature malegametophytes is a widely used plant breeding and propagation technique.

The haploid multicellular male gametophyte of plants, the pollen grain,is a terminally differentiated structure whose function ends atfertilization. Unlike mature gametophyte, the immature gametophyteretains its capacity for totipotent growth when cultured in vitro. Whencultured in vitro an immature gametophyte can be induced to form haploidembryos. This way of forming haploid embryos was described nearly 50years ago, but one that is poorly understood at the mechanistic level.

Haploid embryo development (also referred to as microsporeembryogenesis, pollen embryogenesis or androgenesis) is induced byexposing anthers or isolated gametophytes to abiotic or chemical stressduring in vitro culture (see Touraev, A., et al (1997) Trends Plant Sci.2: 297-302. These stress treatments induce sustained division of thegametophyte leading to the formation of a histodifferentiated haploidembryo.

Brassica napes is one of the most well studied models for microsporeembryogenesis (see Ousters, J. B. M., et al, (2001) Current trends inthe embryology of angiosperms. In: Androgenesis in Brassica, a modelsystem to study the initiation of plant embryogenesis, S. S. Bhojwaniand W. Y. Soh, eds (Dordrecht: Kluwer Academic Publishers), pp.451-470). A heat-stress treatment is used to induce microsporeembryogenesis in this and other Brassica species. Only a smallpercentage of the heat-stressed immature male gametophytes will developinto differentiated embryos, although the number ofsporophytically-dividing cells may be initially much higher.

Microspore-derived embryogenesis is a unique process in which haploid,immature pollen (microspores) are induced by one or more stresstreatments to form embryos in culture. These microspore-derived embryos(MDEs) can be germinated and converted to homozygous doubled haploid(DH) plants by chromosome doubling agents and/or through spontaneousdoubling. DH production is a major tool in plant breeding and traitdiscovery programs as it allows homozygous lines to be produced in asingle generation. This quick route to homozygosity not only drasticallyreduces the breeding period, but also unmasks traits controlled byrecessive alleles. DHs are widely used in crop improvement as parentsfor F1 hybrid seed production, to facilitate backcross conversion, formutation breeding, and to generate immortal populations for molecularmapping studies.

The morphological and cellular changes that occur during the inductionand development of haploid embryos have been well described, howeverthere is still very little known about the mechanisms underlying thisprocess. Molecular screens have been performed to identify the changesthat occur during the induction and growth of haploid embryos, howeverno specific genes or signalling pathways have been unequivocallyidentified as causal factors.

Many years of cell biological studies in model species such as tobacco,barley and Brassica, have laid a solid foundation for understanding thecellular events that accompany haploid embryogenesis, yet the mechanismunderlying this change in developmental pathways is not known (seeMercedes S. et al., (2013) Plant Reprod. 26: 181-196).

Li, W-Z. et al, (2001) In Vitro Cell. Dev. Biol.-Plant 37: 605-608describes the effects of DNA hypomethylating drugs azacytidine andethionine on androgenesis in barley (Hordeum vulgare L.).

Furuta, K., et al. (2011) Plant Cell Physiol. 52: 618-628 is entitled“The CKH2/PKL chromatin remodelling factor negatively regulatescytokinin responses in Arabidopsis calli.” Subject of this scientificwork were two isolated mutants of Arabidopsis thaliana, ckh1 (cytokinehypersensitive 1) and ckh2. These mutants are cytokine hypersensitiveand produce rapidly growing (diploid) green calli in response to lowerlevels of cytokines. The authors were looking for a mechanism behind thecytokinin-inducible callus greening. Trichostatin A (TSA) was found ableto partially replace the growth regulator cytokinin in callus formationfrom hypocotyl segments, which usually requires auxin and cytokinin. Thestarting material and calli tested were all diploid. Such diploid calliare organized, rooty and organogenic.

Acetylation and deacetylation of the lysine residue in histone proteinsare often involved in the reversible modulation of chromatin structurein eukaryotes and can mediate the positive-negative regulation oftranscription. Histone acetyltransferase catalyzes histone acetylation.Histone deacetylase (HDAC) catalyzes histone deacetylation. Hitherto, anumber of disparate and yet putative functions for HDACs in plants havebeen suggested in the scientific literature.

For example, Tanaka M, et al. (2008) Plant Physiol. 146: 149-61 reportson effects of HDAC inhibitor trichostatin A (TSA) on seed germination inArabidopsis. Normally, Arabidopsis seeds show radicle emergence withcotyledon expansion and greening within about 7 days after sowing. Incontrast, following treatment with TSA, most Arabidopsis seeds showradicle emergence, but no cotyledon expansion or greening. This is alsoassociated with expression of embryo-specific factors and the formationof embryo-like structures. A TSA concentration-dependentpost-germination growth arrest was observed, as well as formation ofembryo-like structures after germination. The authors suggest a role forHDAC following germination in the repression of existing embryonicproperties in Arabidopsis, but without indication as to any mechanism.

Although DH production is widely exploited, there are often one or morebottlenecks that need to be overcome before an efficient DH productionsystem can be established for a specific crop or genotype. One majorbottleneck is a low level of haploid embryo induction. Entire speciesare often recalcitrant, and even responsive species show a stronggenotypic component for DH production. In non-responsive genotypes, themicrospores either fail to divide or arrest early in their development.A second bottleneck is the low rate of embryo germination and conversionto plantlets, a phenomenon that has been attributed to poor meristemdevelopment.

Histone deacetylase inhibitors (HDACi) have a long history and have beenused in psychiatry and neurology as mood stabilizers andanti-epileptics. More recently they are being investigated further inrelation to possible treatments for inflammatory diseases and cancers.

DISCLOSURE OF THE INVENTION

The inventors have discovered that by using a chemical approach, theswitch to haploid embryogenesis is controlled by the activity of histonedeacetylases (HDACs). Blocking at least part of HDAC activity with aninhibitor of HDAC (HDACi) in cultured immature male gametophytes leadsto a large increase in the proportion of cells that switch from pollento embryogenic growth. Whilst not wishing to be bound by any particulartheory, the inventors have found that HDACi used in microspore cultureblocks an existing developmental program and causes switch to a newprogram.

The inventors also discovered that HDACi induced embryogenesis andgrowth may be enhanced by, but is not dependent on, other stress, suchas high temperature stress.

The inventors have also discovered that the immature male gametophyte ofa species recalcitrant for haploid embryo development in culture, alsoforms embryogenic cell clusters after HDAC inhibitor treatment.

Accordingly, the present invention provides a method of producinghaploid plant embryos comprising culturing haploid plant material in thepresence of a histone deacetylase inhibitor (HDACi). Culturing mayinclude the growing of haploid material in the presence of HDACi.

The invention also includes a method of producing haploid seedlingscomprising exposing haploid plant material to a histone deacetylaseinhibitor (HDACi) to produce haploid embryos and then converting (i.e.germinating) the haploid embryos into seedlings.

The invention therefore includes a method of making haploid plantscomprising growing a seedling produced in accordance with theaforementioned method.

The invention also provides a method of producing a double haploid plantcomprising culturing haploid plant material in the presence of a histonedeacetylase inhibitor (HDACi) for a period, stimulating or allowing aspontaneous chromosome doubling, and growing the double haploid plantmaterial into a seedling, plantlet or plant.

In certain embodiments, haploid embryogenesis and chromosome doublingmay take place substantially simultaneously. In other embodiments, theremay be a time delay between haploid embryogenesis and chromosomedoubling. The time delay may relate to the developmental stage reachedby the growing haploid embryo, seedling or plantlet. Should growth ofhaploid seedlings, plants or plantlets not involve a spontaneouschromosome doubling event, then a chemical chromosome doubling agent maybe used in accordance with procedures which the average skilled personwill be familiar, see for example:

Various possibilities arise, including exposing haploid plant materialto a histone deacetylase inhibitor (HDACi) until a stage is reachedwhere at least one of: a haploid multicellular globular mass, a globularembryo, a torpedo embryo, an embryo with cotyledon(s) is formed, thengrowing the haploid plant material onwards from that stage in culturefor a period of time to allow a spontaneous chromosome doubling, andregenerating the subsequent double haploid plant material in culture toform a seedling. Where a microspore is exposed to the HDACi, then once asporophytic growth path is identifiable from, for example from one ofthe stages of symmetric division, multicellular globular mass orglobular microspore derived embryo (MDE), heart embryo, torpedo embryoand then embryo with cotyledon(s), then HDACi exposure may be stoppedand the sporophytic growth or embryo growth continued in suitable growthmedium. The growth medium may simply be the same as during HDACiexposure, but without the HDACi present.

Each of the stages of symmetric division, multicellular globular massare readily visualised under a microscope by a person of ordinary skillin this field of art. Similarly, each of the stages of or globularmicrospore derived embryo (MDE), heart embryo, torpedo embryo and thenembryo with cotyledon(s) are readily visualised under a low powermicroscope by a person of ordinary skill in this field of art.

Where a microspore is exposed to the HDACi, then a callus may form andthis may undergo organogenesis to form an embryo. The inventiontherefore includes a method of producing haploid plant callus comprisingexposing haploid plant material to a histone deacetylase inhibitor(HDACi).

Without wishing to be bound by particular theory, the inventors identifytwo potential sporophytic pathways; one which produces compact embryosthat remain enclosed in the exine until between about 5 to 7 days ofculture. The other producing cells that emerge earlier from the exineand show varying degrees of cell connectedness. However, both pathwaysexpress embryo program genes. The invention therefore includes a methodof producing a haploid plant comprising exposing haploid plant materialto a histone deacetylase inhibitor (HDACi) to form a callus andregenerating a plant from the callus.

Without wishing to be bound by particular theory, the inventors believethat a different type of callus is formed after HDACi treatment ofmicrospores than is formed during shoot or root organogenesis. This typeof callus is non-rooty and embryogenic.

As described herein, the term “plant” includes a seedling. A plant mayalso be a plant at any stage of growth and development from seedling tomature plant.

The plant and therefore the plant gametophyte may be an angiosperm or agymnosperm. When an angiosperm, then the plant may be a monocot or adicot.

The exposure of plant material to HDACi is preferably carried out for aperiod of time sufficient to induce haploid embryo formation. Where thestarting haploid material is a microspore, this period of time may bedetermined by the developmental stage reached, e.g. symmetric division,multicellular globular mass or globular microspore derived embryo (MDE),heart embryo, torpedo embryo and then embryo with cotyledon(s). Thestage reached and therefore period of time needed may depend on thespecies of plant concerned and these are all readily ascertainable by aperson of ordinary skill in the art.

Where microspores and subsequent sporophytic developmental stages areexposed to HDACi in accordance with the methods of the invention, thenthis may take place for a period of time or times measured in hours. Forexample, a number of hours in the range 1-24, or 2-24, or 3-24, or 4-24,or 5-24, or 6-24, or 7-24, or 8-24, or 9-24, or 10-24, or 11-24, or12-24, or 13-24, or 14-24, or 15-24, or 16-24, or 17-24, or 18-24, or19-24, or 20-24, or 21-24, or 22-24, or 23-24 hours. Alternatively, anumber of hours in the range 1-23, or 1-22, or 1-21, or 1-20, or 1-19,or 1-18, or 1-17, or 1-16, or 1-15, or 1-14, or 1-13, or 1-12, or 1-11,or 1-10, or 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2hours. A preferred range of HDACi exposure is from about 1 to about 20hours; more preferably from about 2 to about 20 hours.

The period of exposure with HDACi may be measured in terms of days.Though a duration of more than about a day may not necessarily result ingreater frequency of haploid embryo formation, the number of days may bein the range of from about 1 day to about 2 days, about 1 day to about 3days, from about 1 day to about 4 days. A longer number of days than 4may be used if desired.

Once haploid embryos are formed and observable, at whatever desiredstage, then the embryo may be transferred to a growth medium free ofHDACi. The growth medium may be a liquid or a solid medium. The growthmedium will contain all the necessary compounds and factors that arenecessary for the maintenance and/or further growth of the haploidembryo. Generally, the growth medium may be based on standard growthmedia used for diploid embryos or for haploid embryos produced/derivedfrom seed or tissue culture, subject to modification/optimisation ofcomponents for the particular plant species concerned. Modification ofthe composition of growth media is something well within the range ofskill of a person of ordinary skill in the art.

The exposure of haploid plant material to HDACi may be to a singlecompound or a mixture of compounds. One or more different compounds maybe used in combination, whether simultaneously, separately orsequentially.

When maintaining or growing haploid embryos of any stage, there may be aspontaneous doubling of chromosomes leading to production of a doublehaploid seedling. Spontaneous doubling may occur via a variety ofmechanisms.

Often, a double haploid embryo and resultant seedling may be producedfrom a microspore or other stages and/or cells of the gametophyte byusing a chromosome doubling agent; optionally wherein the chromosomedoubling agent is comprised in a gas, solution or a solid and themicrospore, sporophytic microspore stage, haploid embryo, haploid callusor structure is exposed for a period to the chromosome doubling agent.The doubling agent may be used at any time from embryogenesis onwards,right up until the stage of meiosis would occur. So, doubling agents maybe used on whole plant parts, such as shoots or buds, for example.

In some embodiments of the invention, HDACi and chromosome doublingagent may be present together when the plant material is exposed tothem. The specific timing and protocol for chromosome doubling in eachspecies' haploid material is something that the person of average skillin the art may readily ascertain by trial and error.

In certain aspects of the invention, a physical stress is applied to thehaploid plant material prior to its exposure to the HDACi. The physicalstress may be any of temperature, darkness, light or ionizing radiation,for example. The light may be full spectrum sunlight, or one or morefrequencies selected from the visible, infrared or uv spectrum. One ormore physical stresses or combinations of stress may be used prior toexposure to the HDACi compound. The stresses may be continuous orinterrupted (periodic); regular or random over time. When stresses arecombined over time they may be simultaneous (coterminous or partlyoverlapping) or separate.

In preferred methods of the invention the prior physical stress isremoved prior to exposure to the HDACi compound.

The physical stress may be heat, but any other stress treatment such asstarvation or osmotic stress (e.g. mannitol) may be used. Other stresstreatments include n-butanol or ethanol. A combination of stresstreatments may be used whether separately or simultaneously and ifseparately then optionally sequentially. For example when a heattreatment is used it may be a temperature in the range 20° C.-43° C.;possibly in the range 21° C.-34° C. Depending on the species of plantselected, the prior heat treatment may be at 20° C., 21° C., 22° C., 23°C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32°C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40″C, 41°C., 42° C., or 43″C fora period of time.

For any given stress treatment, the period of time may be from about 5minutes to about 5 days, or a period of time selected from about 10minutes to about 4 days, from about 20 minutes to about 3 days, fromabout 30 minutes to about 2 days, from about 1 hour to about 1 day, orfrom about 2 hours to 12 hours.

The haploid plant material to be subjected to the methods and uses ofthe invention is preferably a gametophyte; preferably an immature malegametophyte (i.e. microspore, or vegetative, generative or sperm cellsof the pollen grain). The male gametophyte material may be comprised inan anther and the anther is subject to any of the aforementioned methodsof the invention.

The invention as described herein may also be applied to an immature ormature female gametophyte (i.e. the megaspore and its derivatives,including the egg cell, the polar nuclei, the central cell, thesynergids, the antipodals). The female gametophyte material may becomprised in an ovule and the ovule is subject to any of theaforementioned methods of the invention.

As described herein, a “histone deacetylase inhibitor” (HDACi) ispreferably a compound which is capable of interacting with a histonedeacetylase and inhibiting its enzymatic activity, thereby reducing theability of a histone deacetylase to remove an acetyl group from ahistone. In some preferred embodiments, such reduction of histonedeacetylase activity is at least about 50%, more preferably at leastabout 75%, and still more preferably at least about 90%. In otherpreferred embodiments, histone deacetylase activity is reduced by atleast 95% and more preferably by at least 99%.

The histone deacetylase inhibitor may be any molecule that effects areduction in the activity of a histone deacetylase. This includesproteins, peptides, DNA molecules (including antisense), RNA molecules(including RNAi and antisense) and small molecules. A protein may be anantibody, monoclonal, polyclonal or chimeric; and a peptide may be afragment of such an antibody.

HDACi compounds suitable for use in accordance with any of theaforementioned methods and uses of the invention in all its aspects arewell known and generally available from commercial sources. Theseinclude the following classes of compound: hydroxamic acids, cyclictetrapeptides, aliphatic acids, benzamides, polyphenolics orelectrophilic ketones. More detailed information about HDACi compoundsis provided in the detailed description below.

In preferred aspects, the method and uses of the invention employ HDACiwhich is trichostatin A (TSA), butyric acid, a butyrate salt, potassiumbutyrate, sodium butyrate, ammonium butyrate, lithium butyrate,phenylbutyrate, sodium phenylbutyrate or sodium n-butyrate.

In certain preferred methods and uses, the HDACi is suberoylanilidehydroxamic acid (SAHA) and this advantageously improves the conversion(i.e. “germination”) of haploid embryos or doubled haploid embryos intoseedlings.

The methods of the invention are particularly suited to achievingimproved haploid embryogenesis than methods involving physical stressalone. For example, subject to the species concerned, when microsporesare subjected to a method of the invention and compared to a controlwhere no HDACi is present, at least 10% more haploid embryos are formed.In certain species this may be at least 25% more, at least 50% more, atleast 75% more, at least 100% more, or at least 200% more. In somespecies the number of haploid embryos may be more than 25% more, morethan 50% more, more than 75% more, more than 100% more or more than 200%more. Plants where increased haploid embryo formation is of particularbenefit are model systems of rapeseed (Brassica napus), tobacco(Nicotiana tabacum), barley (Hordeum vulgare) and wheat (Triticumaestivum).

The methods of the invention are also particularly suited to producinghaploid embryos where this has not been possible successfully so far,whether scientifically or commercially. Methods of the invention may beapplied particularly to such previously recalcitrant species such as aspecies or variety of a genus selected from Arabidopsis, e.g. A.thaliana, or Solanum, e.g. S. esculentum.

The invention also includes a histone deacetylase inhibitor (HDACi) ashereinbefore described, for use in haploid plant embryogenesis; i.e.generating haploid embryos from haploid plant material by exposing thehaploid plant material to HDACi and/or growing the haploid plantmaterial in the presence of HDACi.

Also, the invention includes a histone deacetylase inhibitor (HDACi) foruse in producing double haploid plant material; particularly seedlingswhich are then grown on to form plantlets or plants. Such double haploidplant material is generated in part as a result of haploid plantmaterial undergoing an embryogenic event due to exposure to and/orgrowth in presence of an HDACi.

The invention also provides a kit for performing a method of haploidembryogenesis in plants comprising a first container which includes ahistone deacetylase inhibitor (HDACI) and a second container whichincludes a chromosome doubling agent. Such kits may include a set ofinstructions for using the HDACI and chromosome doubling agents. Eitheror both of the HDACI and doubling agents may be in a concentrated formand require dilution prior to use. The kit may further comprisesolutions for the dilution of the HDACi and/or doubling agent stocksolutions that the kit provides. The HDACi and/or doubling agents may beprovided in dry form and solutions may be provided in the kit for makingup solutions. The kit may be designed for use with a particular plantspecies material and include specific instructions.

The invention will now be described in more detail including by way ofexamples and with reference to the drawings in which:

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a bar chart of results showing the effect of the duration ofTSA treatment on sporophytic cell division in B. napus DH12075microspore culture.

FIG. 2 panels A-F are micrographs of gametophytic (A-C) and sporophyticstructures from B. napus microspore culture, as described in Example 1.Panels G and H are bar charts of data showing the percentage ofdifferent cell types observed in control (G) and TSA-treated cultures(H) at different times, also as described in Example 1.

FIG. 3 panel A is a bar chart showing the effect of TSA on sporophyticgrowth in B. napus microspore culture as described in Example 2. PanelsB G are micrographs of type I-IV sporophytic structures after five (B-E)and 15 (F-G) days of culture, as described in Example 2.

FIG. 4 shows a series of bar charts (A-H) with data showing the effectof TSA and culture temperature on cell division and embryo formation inB. napus microspore culture, as described in Example 2.

FIG. 5 shows micrographs of embryo-expressed GFP reporters in B. napusmicrospore culture, as described in Example 3.

FIG. 6 shows micrographs of five day-old anther cultures, as describedin Example 4.

FIG. 7 shows bar charts (A, D) and micrographs (B, C) showing behaviourof hda and rbr mutants in Arabidopsis anther culture, as described inExample 5.

FIG. 8 is a photograph of Western blots as described in Example 6.

FIG. 9 shows data of the effects of various HDACi on sporophytic celldivision in microspore cultures of B. napus DH 12075; at three differentconcentrations compared to DMSO control.

FIG. 10 shows data of the effects of HDACi on embryo yield in microsporecultures of B. napus DH 12075.

FIG. 11 shows data demonstrating how HDACi improve embryo quality inolder stages of donor pollen.

FIG. 12 shows data demonstrating how HDACi-treated embryos can bereadily converted to seedlings.

FIG. 13 shows data exemplifying how genotype influence degree of TSAenhanced embryogenesis in Brassica rapa.

FIG. 14 consists of micrographs showing the effect to TSA-treated (ii)and control (i) microscope culture embryo development in a recalcitrant(non-responsive) genotype of Brassica oleracea Gongylodes group(kohlrabi), (iii) is an enlargement of a large embryo at 18 days.

FIG. 15 shows data on percentage of embryogenic microspores in 10day-old control and TSA-treated Capsicum annuum microscope cultures.

FIG. 16 shows data a number of embryos obtained per bud used in 45 daysday-old control and TSA-treated Capsicum annuum microscope cultures.

DETAILED DESCRIPTION

The inventors have found that chemical inhibition of HDAC activity usingtrichostatin A (TSA) induces massive cell proliferation in the immaturemale gametophyte of Brassica napus, even in the absence of the heatstress treatment that is usually used to induce haploid embryogenesis.Using cell fate markers, the inventors have shown that the multicellularstructures that develop after ISA treatment are embryogenic, but thatmost of these structures fail to form histodifferentiated embryos.Nonetheless, a higher embryo yield can be obtained after TSA treatmentcompared to untreated controls. TSA treatment is associated withincreased acetylation of histones H3 and H4. Transcriptome analysissuggests that activation of cell cycle-, auxin signalling-, cell wallmobilisation- and embryo gene expression pathways contribute to theobserved phenotypes.

Using a chemical approach, the inventors have found that the switch tohaploid embryogenesis is controlled by the activity of histonedeacetylases (HDACs), Blocking HDAC activity with HDAC inhibitors, e.g.TSA, in Brassica napus, B. rapa, Arabidopsis thaliana and Capsicumannuum male gametophytes leads to a large increase in the proportion ofcells that undergo embryogenic growth. In B. napus, treatment with onespecific HDACi (SAHA) improves the conversion (i.e. germination) ofthese embryos into seedlings.

The inventor's discovery of the utility of HDAC inhibitors for haploidembryogenesis can be used to produce and propagate new plant varieties,but will not be directly incorporated as traits per se in plants. Forplant varieties in which DH production is possible, but inefficient, theinvention will significantly increase the efficiency and decrease thecost of DH production, but will not have a significant impact on thecost of breeding new plants. The main value to be gained for these cropslies in the increased number of new DH lines or crosses from a breedingprogram that can be generated. All tested species so far react in thesame way and so the present invention is also generically applicable,including to those plant species or varieties where DH production hasnot yet been achieved. Advantageously, this avoids having to developtailor-made approaches for each crop/variety.

The inventors have shown that inhibition of histone deacetylation issufficient to induce haploid embryo development in cultured pollen ofboth B, napus and Arabidopsis. Many different stressors can be used toinduce haploid embryogenesis. In this respect, the deregulation of HDACsby stress and the accompanying changes in histone acetylation statusprovides a single, common regulation point for the induction of haploidembryogenesis.

The developmental stage of the vegetative cell plays a major role in itsresponsiveness to stress and TSA. In the majority of species, the stresstreatment is most effective in triggering sustained cell division inculture shortly before or after PM I (Touraev et at (1997) supra).Heat-stressed B. napus microspores can be induced to dividesporophytically when they are at the G1 to G2 phase of the cell cycle,while the vegetative cell of the binucleate pollen is responsive, albeitat a much lower frequency, at G1 (Binarova, P., et al. (1993) Theor.Appl. Genet. 87: 9-16). During normal pollen development the vegetativecell does not divide after PM I and is assumed to arrest in G1 (G0).This stage of pollen development is much less responsive for haploidembryo induction. Unlike heat stress alone, TSA, alone or in combinationwith heat-stress, is highly effective at this late stage of pollendevelopment, and has a much stronger effect than heat-stress alone withrespect to the proportion of cells that divide sporophytically. TSA is amore potent inducer of sporophytic growth due to its ability to morecompletely inhibit individual HDACs or to inhibit a wider range of HDACsthan heat-stress alone. The inventors have found that a relatively highconcentration of TSA in combination with heat stress enhances divisionsthat mainly result in disorganized embryogenic structures, while arelatively low concentration of TSA in combination with heat-stress moreclosely mimics the effect of heat-stress alone in that the formation ofboth histodifferentiated embryos and non-viable disorganized embryogenicstructures is enhanced.

Culture at lower temperatures dampens the effect of TSA, such that fewercells divide, and a higher concentration of ISA is needed to induceembryo and embryogenic cell formation than at 33° C. In line with thisobservation, in B. napus a more severe, 41° C. heat-stress required toinduce sporophytic divisions and embryogenesis at the late bicellularstage (Binarova, P., et al. (1997) Sex. Plant Reprod. 10: 200-208).HDACs (directly or indirectly) mediate the inhibition of cell cycleprogression that is gradually imposed on the vegetative cell, and thatrelease of this inhibition is required for embryogenic growth inculture.

The invention provides tools that can be immediately and easily appliedby plant breeders in a GMO-free manner. The ability to use smallcompounds to improve tissue culture responses eliminates the need tocreate and market transgenic plants, allowing rapid and cost-effectiveinnovation. This is important in the food sector, where consumers arehesitant about consuming transgenic products. A non-transgenic approachis also important when companies have crops/varieties with a smallmarket share, for which the costs involved in developing and marketingtransgenic plants are prohibitive.

A way of determining whether a compound is an HDACi for use inaccordance with any of the aspects or embodiments of the invention is byusing standard enzymatic assays derived from measuring the ability of anagent to inhibit catalytic conversion of a substance by the subjectprotein. In this manner, inhibitors of the enzymatic activity of histonedeacetylase proteins can be identified (see Yoshida, et at, J. BiolChem. 265: 17174-17179 (1990)).

More particularly, an HDACi for use in accordance with any of theaspects or embodiments of the invention described herein includes:trichostatin A (TSA) and compounds related to TSA, such as butyric acid,butyrate salts such as potassium butyrate, sodium butyrate, ammoniumbutyrate, lithium butyrate, phenylbutyrate, sodium phenylbutyrate(NaPBA); also sodium n-butyrate. Also, M344 which is an amide analog ofTSA and analogues disclosed in US2011/0237832.

HDACi compounds for stimulating haploid embryogenesis in accordance withthe invention include: suberoyl bis-hydroxamic acid (SBHA), vorinostat(suberoylanilide hydroxamic acid (SAHA)); valproic acid sodium salt(sodium valproate); Scriptaid (6-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide) (see U.S. Pat.No. 6,544,957).

Also, rocilinostat (ACY-1215); etinostat (MS-275); mocetinostat(MGCD0103, MG0103); belinostat (PXD101); dacinostat (LAQ824);droxinostat (CMH, 5809354); resminostat (RAS2410); panobinostat(LBH589); pracinostat (SB939); givinostat (ITF2357); quisinostat(JNJ-26481585); abexinostat (PCI-24781).

Additionally, Trapoxin; specifically trapoxin A(Cyclo((S)-phenylalanyl-(S)-phenylalanyl-(R)-pipecolinyl-(2S,9S)-2-amino-8-oxo-9,10-epoxydecanoyl)and cyclic tetrapeptide compounds related to trapoxin A having the aminoacid-2-amino-8oxo-9,10-epoxy-decanoic acid in their molecules, e.g.chlamydocin (Closse, et al., Helv. Chim. Acta 57: 533-545 (1974)),HC-toxin (Liesch, et al., Tetrahedron 38: 45-48 (1982)); Cyl-2; andWF-3161 (Umehara, K. J. Antibiot 36: 478-483 (1983). Trapoxin B may beused.

The following HDACi compounds are also suitable for use in accordancewith the invention: oxamflatin((2E)-5-[3-(Phenylsulfonylamino)phenyl]-pent-2-en-4-ynohydroxamic acid);depsipeptides such as romidepsin and spiruchostatin A; hybrid polarcompounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA) andm-carboxycinnamic acid bishydroxamide (CBHA); apicidin(CycloR2S)-2-amino-8-oxodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperidinexcarbonyl]);depudecin(4,5:8,9-Dianhydro-1,2,6,7,11-pentadeoxy-D-threo-D-ido-undeca-1,6-dienitol);romidepsin; trapoxin; radicicol; cambinol5-(2-Hydroxynaphthalen-1-ylmethyl)-6-phenyl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one;tubacin; tubastatin A HCl; resveratrol 3,4′,5-Trihydroxy-trans-stilbene;splitomicin 1,2-Dihydro-3H-naphtho[2,1-b]pyran-3-one; tacedinaline(C1994); sulindac; PXD101; PTACHS-[6-(4-Phenyl-2-thiazolylcarbamoyl)hexyl] thioisobutyrate; CUDC 101(7-[[4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-yl]oxy]-N-hydroxyheptanamide);MOCPAC (Benzyl(S)-[1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5-propionylaminopentyl]carbamate): MC1568; PCI-34051; C1-994 (:4-Acetylamino-N-(2′-aminophenyl)benzamide); CUDC-101; CUDC-907; LAQ 824;AR-42 (OSU-HDAC42); APHA Compound 8(3-(1-Methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide);BATCP(5)-[5-Acetylamino-1-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl)pentyl]carbamicacid tert-butyl ester; MGDCD0103; 8B939; CHR-2845; CHR-3996; 480-202;Sulforaphane; Kevetrin.

Amongst polyphenolic HDACi compounds, naturally occurring plantpolyphenols having this activity may be used. For example,(−)-epigallocatechin-3-gallate (EGCG) and genistein (GEN) as well asoxidative methyleugenol (ME) metabolites.

Natural products with HDACi activity are available and may be used inaccordance with the invention, including: curcumin, butyrate, diallyldisulphide, sulfopropane and parthenolide.

Other HDACi molecules may include proteins and peptides, includingantibodies or fragments thereof, preferably monoclonal antibodies thatspecifically react with the histone deacetylase.

While the concentration range of the HDACi used will vary and willdepend on the specific inhibitor. The concentration range may thereforebe from about 0.001 nM to about 100 mM; preferably a range selected fromone of the following: from about 0.01 nM to about 50 mM; from about 0.05nM to about 10 mM; from about 0.1 nM to about 5 mM; from about 0.5 nM toabout 1 mM; from about 1 nM to about 500 μM; from about 5 nM to about250 μM; from about 10 nM to about 100 μM; from about 25 nM to about 50μM.

Where artificial chromosome doubling is required in accordance withaspects and embodiments of the invention, suitable methods are taught inAntoine-Michard, S. et al., (1997) Plant cell, tissue organ cult.,Cordrecht, the Netherlands, Kluwer Academic Publishers, 48(3): 203-207;Kato, A., Maize Genetics Cooperation Newsletter (1997) 36-37; and Wan,Y. et al., TAG (1989) 77: 889-892; and Wan, Y. et al., TAG (1991) 81:205-211. Additional technical guidance for chromosome doubling isprovided by Segui-Simarro J. M., & Nuez F. (2008) Cytogenet. Genome Res.120: 358-369. Many procedures involve contact of plant cells withcolchicine, anti-microtubule agents or anti-microtubule herbicides suchas pronamide, nitrous oxide, or any mitotic inhibitor. The result ishomozygous doubled haploid cells.

Where colchicine is used, the concentration in the medium may begenerally 0.01%-0.2% or approximately 0.05% or APM (5-225 μM). The rangeof colchicine concentration may be from about 400-600 mg/L or about 500mg/L.

Where pronamide is used the medium concentration may be about 0.5-20 μM.Examples of known mitotic inhibitors are listed below. Other agents suchas DMSO, adjuvants or surfactants may be used with the mitoticinhibitors to improve doubling efficiency.

Common or trade names of suitable chromosome doubling agents include;colchicine, acetyltrimethylcolchicinic acid derivatives, carbetamide,chloropropham, propham, pronamide/propyzamide tebutam, chlorthaldimethyl (DCPA), Dicamba/dianat/disugran (dicamba-methyl) (BANVEL,CLARITY), benfluralin/benefin/(BALAN), butralin, chloralin, dinitramine,ethalfluralin (Sonalan), fluchloralin, isopropalin, methalpropalin,nitralin, oryzalin (SURFLAN), pendimethalin, (PROWL), prodiamine,profluralin, trifluralin (TREFLAN, TRIFIC, TRILLIN), AMP (Amiprofosmethyl); amiprophos-methyl Butamifos, Dithiopyr and Thiazopyr.

The chromosome doubling agent may be contacted with an haploid embryo atvarious times. If the embryo is isolated the doubling agent may come incontact immediately after isolation. The duration of contact between thechromosomal doubling agent may vary. Contact may be from less than 24hours, for example 4-12 hours, to about a week. The duration of contactis generally from about 24 hours to 2 days.

The invention is applicable to any angiosperm plant species, whethermonocot or dicot.

Preferably, plants which may be subject to the methods and uses of thepresent invention are crop plants such as cereals and pulses, maize,wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea,and other root, tuber or seed crops. Important seed crops are oil-seedrape, sugar beet, maize, sunflower, soybean, and sorghum. Other plantsto which the present invention may be applied may include lettuce,endive, and vegetable brassicas including cabbage, broccoli, andcauliflower, and carnations, geraniums, tobacco, cucurbits, carrot,strawberry, sunflower, tomato, pepper, chrysanthemum.

Grain plants that provide seeds of interest and to which methods anduses of the invention can be applied include oil-seed plants andleguminous plants. These include grain seeds, such as corn, wheat,barley, rice, sorghum, rye, etc. Oil-seed plants include cotton,soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut,etc. Leguminous plants include beans and peas. Beans include guar,locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, limabean, fava bean, lentils and chickpea.

In particular, the invention is applicable to crop plants such as thoseincluding: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.),alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerate),sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthusannua), wheat (Triticum aestivum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachishypogaea), cotton (Gossypium hirsutum), sweet potato (Iopmoea batatus),cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocosnucifera), pineapple (Ananas comosus), citrus tree (Citrus spp.) cocoa(Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado(Persea americana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew(Anacardium occidentale), macadamia (Macadamia intergrifolia), almond(Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley,vegetables and ornamentals.

Similarly, the invention can be applied to perennial fast growingherbaceous and woody plants, for example trees, shrubs and grasses. Anon-exhaustive list of examples of tree types that can be subjected tothe methods and uses of the invention includes poplar, hybrid poplar,willow, silver maple, black locust, sycamore, sweetgum and eucalyptus.Shrubs include tobacco. Perennial grasses include switchgrass, reedcanary grass, prairie cordgrass, tropical grasses, Brachypodiumdistachyon, and Miscanthes.

DH production is a major trait discovery and breeding tool, as describedabove. The HDACi compounds can be used to overcome two major bottlenecksin haploid embryo culture: induction of embryogenic divisions/embryosand conversion of embryos to seedlings.

The current best mode of the invention is a use of SAHA in Brassicanapus microspores to achieve increased haploid embryogenesis andimproved conversion of embryos into double haploid seedlings.

The inventors have also succeeded in achieving increased embryogenicdivisions in immature male gametophytes of Brassica rapa and Capsicumannuum when exposing them to TSA

In the description of experimental examples of the invention whichfollows, the following materials and methods were employed.

Plant Material and Culture

Brassica napus L. cv. Topas DH4079 and DH12075 were used as donor plantsfor microspore embryo culture. The B. napus plant growth and microsporeisolation procedures were performed as described in Ousters, J. B. M.(2003) “Microspore culture in rapeseed (Brassica napus L.)” in: Doubledhaploid production in crop plants: a manual, M. Maluszynski, K. J.Kasha, B. P. Forster, and I. Szarejko, eds (Dordrecht: Kluwer AcademicPublishers), pp. 185-193. Flower buds for microspore culture weregrouped by size (measured from the tip of the flower bud to the bottomof the sepal), ranging from 3.0 to 3.5 mm for DH4079 and from 2.6 to 4.0mm for DH12075. The microspores were isolated and cultured in NLN-13medium (see Lichter, R. (1982) Mel. Plant 3: 594-602. For induction ofembryogenesis, microspores were cultured in the dark at 33° C. for 20hours, and subsequently transferred to 25° C. Non-induced microsporecultures were cultured continuously at 25° C. or 18° C. Trichostatin A(TSA, Sigma-Aldrich) was prepared in DMSO. Freshly isolated microsporeswere inoculated in medium containing TSA or the same volume of DMSO as acontrol. and cultured for 20 hours at the temperature indicated for eachexperiment. After this period the cultures were centrifuged at 200 g for3 min, resuspended in fresh NLN-13 medium without TSA, and transferredto 25° C.

Arabidopsis flower buds at stage 11 were collected for anther culture.Flower buds were surface sterilized in 2% bleach for 10 minutes, thenrinsed three times in distilled water. The anthers (without filament)were placed in liquid NLN-13 medium containing 0.5 μM ISA or the samevolume of DMSO, and then cut in half transversely in the medium torelease the microspores. The cultures were placed at 25° C. for 20 hoursin the dark. The medium was then replaced by fresh NLN-13 medium bypipetting gently, and the cultures incubated at 25° C. for an additionalfour days. Free and loosely attached microspores were collected andstained with DAPI. Arabidopsis tide T-DNA insertion lines were obtainedfrom Nottingham Arabidopsis Stock Centre. At least 300 microspores persample were counted.

Reporter Lines

GFP-based reporter lines were generated for the Arabidopsisembryo-expressed genes, LEC1 (At1g21970; LEC1:LEC1-GFP) and GRP(At2g30560; GRP:GFP-GUS) and the B. napus ENODL4 gene (AB836663;ENODL4:GFP). For the LEC1:LEC1-GFP translational fusion, a 3110 bp DNAfragment comprising 1292 bp upstream of the translational start site andthe entire coding region was amplified by PCR and recombined into pGKGWGusing the Gateway cloning system (Invitrogen) according to themanufacturer's instructions. The Arabidopsis GRP gene encodes an EGGAPPARATUS1-LIKE (EAL) protein (see Gray-Mitsumune, M., and Matton, D. P.(2006) Planta 223: 618-625) and is highly similar to a B. napusglycine-/proline-rich gene isolated from embryogenic microspore cultures(probe 563; see Joosen, R., et al, (2007) Plant Physiol. 144: 155-172).The Arabidopsis GRP:GFP-GUS transcriptional fusion was made by PCRamplifying a fragment comprising 861 bp upstream of the start codon andGateway recombination into pBGWFS7,0. The BnENODL4 was identified as anearly embryogenesis-expressed gene from B, napus microspore culture(Japanese Patent No. 35935650). A 1035 bp fragment of the promoter ofBnENODL4 gene (GenBank accession no. AB098076) was cloned by inversePCR, ligated to the 5′-end of an sGFP: nos terminator fragment andinserted into pBinKH, which is a modified version of a binary vectorpGPTV-KAN (see Becker, D., et al. (1992) Plant Mol. Biol. 20:1195-1197). The reporter constructs were transformed to Agrobacteriumtumefaciens strain C5801 carrying the pMP90 Ti plasmid and then to B.napus DH12075 (see Moloney, M. M. et al. (1989) Plant Cell Rep. 8:238-242) and/or Arabidopsis 0010 (see Clough, S. J., and Bent, A. F.(1998) Plant J. 16: 735-743).

Microscopy

The developmental stage and identity of cells in microspore and antherculture were visualized with the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI, 1.25 μg/ml according to Ousters (2003)supra using a Zeiss Axioskop epifluorescence microscope with filter setno. 02. Approximately two hundred microspores or multicellular clusterswere counted for each sample. GFP was imaged using confocal laserscanning microscopy (CLSM; Leica DM5500 Q). The GFP was excited with anargon laser line at 488 nm and detected with a 505-530 nm emissionfilter. Samples were counterstained with DAPI or propidium iodide (10mg/ml; Sigma-Aldrich), Propidium iodide and red autofluorescence wereexcited at 532 nm and detected with a 620-660 nm emission filter. GFPand DAPI were covisualized with CLSM, For CLSM, DAPI was excited at 405nm and detected with a 440-500 nm emission filter. The optical sliceswere median filtered with Leica LAS AF software. Arabidopsis antherswere cleared in HOG solution (water: Chloral hydrate: glycerol; 3:8:1)for 10 min, then observed under DIC microscopy with a Nikon OPTIPHOTmicroscope.

Molecular Analyses

Total RNA isolation and on-column DNase digestion were performed usingthe InviTrap Spin Plant RNA Mini Kit (Invitek) according to themanufacturer's instructions. For semi-quantitative RT-PCR, 250 ng oftotal RNA was used for first-strand cDNA synthesis with the TaqmanReverse Transcription Reagents Kit (Applied Biosystems). The cyclingparameters were: one cycle at 98° C. for 30 s, 30 cycles comprising 98°C. for 5 s, 60° C. for 30 s, followed by 72° C. for 1 min. Thesemi-quantitative RT-PCR primers are from Malik et al. (2007) PlantPhysiol. 144: 134-154. The quantitative RT-PCR primers for microarrayvalidation were designed based on oligonucleotide probes from AffymetrixGeneChip® Brassica Exon 1.0ST Array (see Malik et al, (2007) supra andLove, C. G., et al. (2010) PloS one 5: e12812). The Arabidopsis hdaT-DNA insertion lines were genotyped using the PCR primers. Microsporecultures for microarray analysis were cultured at 33° C. for eight hourswith either TSA, cycloheximide (CHX, Sigma-Aldrich) dissolved in DMSO,DMSO or cycloheximide, or with TSA and cycloheximide together. Thesamples were harvested by centrifugation for total RNA was isolation, asdescribed above. One microgram of total RNA from each sample was sent tothe NASC Affymetrix Service for hybridisation to the Affymetrix BrassicaExon 1.0 ST GeneChip. Probe annotations were downloaded from GeneExpression Omnibus. The identifier for the annotation is GPL10733. Theexpression data was subjected to normalization using the RMA method fromthe ‘affy’ Bioconductor package. Log 2-transformed expression valueswere identified as differentially expressed using a Student's t-test.Multiple hypothesis testing correction was done using the Holm's method(Holm, S. (1979) Scandinavian Journal of Statistics 6: 65-70)implemented in the multtest's Bioconductor package. Mapman (see Thimm,O., et al. (2004) Plant J. 37: 914-939 was used to identify functionalcategories of differentially-expressed genes. The microarray data hasbeen deposited to the Gene Expression Omnibus (GEO) database (GSE49070).

Immunochemistry

Freshly isolated microspores and microspores cultured for 8 hours underdifferent experimental conditions were harvested by centrifugation.Proteins were extracted by boiling in SDS-sample buffer (30 μl per ml ofculture) and electrophoresed in a Midget 12.5% SDS-PAGE gel underreducing conditions. After transfer of the proteins to PVDF membrane andblocking with 5% milk powder in PBS, 0.1% Tween 20, the blots wereincubated for 2 hours with primary antibody (1:2000 dilution). Theprimary antibodies used in this study are as follows: anti-acetyl-Lysine(ICP0380; ImmuneChem Pharmaceuticals), anti-Histone H3 (ab1791; Abcam),anti-Histone H4 (clone 62-141-13; Millipore), and anti-acetyl-Histone H3and anti-acetyl-Histone H4 (Millipore). Secondary goat anti-rabbit-HRPantibody (Sigma) was used in a 1:2000 dilution and signals were detectedby using enhanced chemiluminescence (SuperSignal West FemtoChemiluminescent Substrate, Pierce).

Example 1—TSA Induces Hyperproliferation in Poorly Responsive B. napusGenotype, DH12075

Cultured microspores and pollen of B. napus genotype, DH12075 weretreated with the HDAC inhibitor, TSA. We examined the development ofmicrospore cultures by staining heat-stressed (hereafter referred to ascontrol) and heat-stressed plus TSA-treated immature male gametophytesat different developmental stages with the nuclear dye, DAPI. Initialdosage experiments were used to establish the minimal exposure time (20h) in relation to the specific phenotypes discussed below.

FIG. 1. Effect of the duration of TSA treatment on sporophytic celldivision in B. napus DH12075 microspore culture. Immature malegametophytes from two different bud sizes (black bars and white bars)were cultured in the presence of 0.5 μM TSA or with the equivalentvolume of DMSO (control) at 33° C. Sporophytic cell divisions werecounted after DAPI staining after five days of culture. Treatments forlonger than 20 hours did not further enhance or reduce the proportion ofsporophytic divisions.

FIG. 2 shows the effect of TSA on early cell division patterns in B.napus microspore culture. DAPI-stained gametophytic (A-C) andsporophytic structures (D-F) are present in the first two days ofmicrospore culture, (A)=microspore, (B)=binucleate pollen,(C)=trinucleate pollen, (D)=sporophytically-divided cell with twodiffusely-stained vegetative-like nuclei. (E)=sporophytic structure withthree vegetative-like nuclei and one condensed, generative-like nucleus.(F)=multinucleate sporophytic structure with four vegetative-like nucleiand two generative-like nuclei, (G-H)=the percentage of different celltypes observed in control (G) and TSA-treated cultures (H). The celltypes were grouped into the following categories: dead gametophytes(white bars); gametophytic structures at the microspore (light greybars); binucleate (medium grey bars) and trinucleate (dark grey bars)stages; and sporophytically-divided structures (black bars). Control,DMSO treated sample. Immature male gametophytes were obtained from donorflower buds that were grouped by size. The samples are ranked fromyoungest to oldest (1-6) based on the developmental stages of the malegametophytes found in each donor bud size group. V=vegetative(-like)nucleus; g=generative(-like) nucleus; s=sperm nucleus. Scale bar=10 μm.

After two days of heat stress, immature male gametophytes in controlcultures arrest, continue pollen development, or divide sporophytically.Male gametophyte development in culture follows the same course ofdevelopment as in the anther (see FIGS. 1A-C). The single-celledmicrospore divides asymmetrically (pollen mitosis I, PM I) to generate apollen grain with a large vegetative cell containing a diffuse nucleusand a smaller generative cell with a more compact nucleus. Thevegetative cell arrests in GIGO, while the generative cell divides oncemore (pollen mitosis II, PM II) to produce the two gametes, the spermcells, that participate in double fertilisation. Microspores that dividesporophytically contain two large, diffusely-stained nuclei, rather thanthe large, diffusely-stained vegetative nucleus and small condensedgenerative nucleus produced after PM I (FIG. 2D). Immature gametophytesthat divide sporophytically after PM I, which is rarely (<1%) observedin control cultures from this genotype, contain a small generative-likecell in addition to the larger sporophytic cells (FIG. 2E). After heatstress treatment, the majority of the cells in the control culture weregametophytic-like or had died, as evidenced by the lack of DAPI staining(FIG. 2G). Approximately 6% of the population divided sporophytically inthe first two days of cultures, producing cell clusters with two to sixnuclei. The developmental stage of the starting population in thecontrol cultures did not influence the initial proportion of cells thatdivided sporophytically.

The combined effect of heat stress and 0.5 μM TSA on sporophytic celldivision after two days of culture was dramatic, with up to 80% of thepopulation dividing sporophytically (FIG. 2H). The largest increase inthe proportion of sporophytically-divided structures was observed incultures that initially contained binucleate pollen. The majority ofsporophytically-divided cells cultures contained two to sixdiffusely-stained nuclei, as in control cultures. Unlike controlcultures, approximately 10% of the sporophytically-divided cells alsocontained one or more generative-like nuclei (FIG. 2F). The lowfrequency of cells with generative-like nuclei is surprising consideringthe 40 to 60% binucleate pollen that was present at the start ofculture. The generative nucleus may degrade, or may assume a morediffuse morphology, perhaps contributing to the observed ectopicdivisions.

The observations indicate that loss of HDAC activity in culturedimmature male gametophytes induces a high frequency of ectopicsporophytic cell division. HDAC proteins appear to play a major role incontrolling cell cycle progression during male gametophyte development.The combined effect of heat-stress and TSA treatment is more potent thanthat of heat-stress alone, both in terms of the developmental stages andthe proportion of immature gametophytes that are induced to dividesporophytically.

Example 2—TSA and Heat-Stress Induce Similar Developmental Changes

The developmental fate of heat-stressed control cultures and culturesexposed to both heat-stress and ISA was followed by examining oldercultures in more detail. Initial experiments showed that the proportionof dividing cells, as well as their developmental fate was influenced bythe concentration of TSA that was applied to the culture. Heat-stressedmicrospores and pollen were treated with a range of TSA concentrationsand the cultures examined after five and 15 days using DAPI staining tocharacterize the different multicellular structures that developed.

FIG. 3 shows the effect of TSA on sporophytic growth in B. napusmicrospore culture. (A)=percentage of cells that had dividedgametophytically (white bars) or sporophytically (grey bars) after fivedays of microspore culture. The corresponding structures are shown inFIG. 3 B-E (has scale bar of 20 μm) where FIG. 3(B-G) shows images oftype I-IV sporophytic structures after five (B-E) and 15 (F-G) days ofculture. The sporophytic cell clusters are categorized as follows: TypeI, classical embryo-forming structures (black bars, FIG. 3B); Type II,compact callus-like structures (dark grey bars, FIG. 3C); Type extrudedsporophytic structures (medium grey, FIG. 3D) and Type IV, loosecallus-like structures (light grey bars, FIG. 3E), Type II structure(FIG. 3F) and Type IV structure (FIG. 3G). Dead microspores and pollenwere not included. Control is a DMSO treated sample. Nuclei in FiguresB-G are stained with DAPI. Arrow shows intact (B) or broken (C, D, E, F)exine. The developmental stages of the starting material (1-8) areranked from youngest to oldest.

Four types of sporophytic structures were distinguished in five-day oldcontrol cultures (FIG. 3B-E), some of which have been previouslydescribed in microspore cultures of other Brassica genotypes. Type 1structures are the classical embryo-forming structures that areroutinely observed in microspore culture. After five days of culturethese multicellular structures contained up to 40 nuclei that were stillenclosed in the pollen wall (exine; FIG. 3B). Cell walls were formed inType I structures, but were not clearly visible. These embryogenicmulticellular structures were only observed in control cultures thatinitially contained a mixture of late uninucleate microspores and earlybinucleate pollen; and only comprised a small proportion of thepopulation of dividing cells (0,5%), Type II structures were the mostabundant structures present in five day control cultures. They arecallus-like; less compact than Type I structures, and contain up to fivecells that had already started to emerge from the exine (FIG. 3C). TypeIII structures contained two to three large and diffusely-stained nucleiand were no longer enclosed by the exine, which remained attached to thecell clusters and was often associated with a generative-like nucleus(FIG. 3D), Type IV structures, which were rarely observed in controlcultures, comprised loose callus-like clusters with DAPI stained cellwalls (FIG. 3E).

The same sporophytic structures as in the control were observed in fiveday old cultures that received a combined heat-stress and TSA-treatment,but in different proportions depending on the concentration of TSA thatwas applied (FIG. 3A).

FIG. 4 shows the effect of ISA and culture temperature on cell divisionand embryo formation in B. napus microspore culture. Microspores andimmature pollen from different bud sizes were cultured in the presenceof different concentrations of ISA compared with the equivalent volumeof DMSO (control) at three temperatures: A−D=33° C., D−F=25° C., G−H=18°C. For each treatment, the samples are ranked from left to right alongthe z-axis according to the developmental stage of the microspores andpollen, A, D, G shows developmental stage of microspores and pollen atthe start of culture in the experiments in A=33° C., D=25° C. and G=18°C. The microspores and pollen were categorized as follows:mid-uninucleate microspore (white bars); late-uninucleate microspore(grey bars) and binucleate pollen (black bars) stages. B, E, H shows theeffect of TSA on cell division in B. napus microspore embryo culture.The percentage of cells that divided gametopyhytically orsporophytically after five days of microspore culture at B=33° C., E=25°C., H=18° C. The sporophytic cell clusters were examined after five daysof culture and categorized as follows: Type I, classic embryogenicstructures (black bars); Type II, compact callus-like structure (darkgrey bars); Type III, extruded sporophytic structures (medium grey bars)and Type IV, loose callus-like structure (light grey bars). Gametophyticcell types are indicated by white bars. Dead microspores and pollen werenot counted. C, F show embryo yield from microspores and pollen formedin the presence of DMSO (control) or different concentrations of TSA atC=33 CC or F=25° C. Histodifferentiated embryos did not develop incontrol and TSA-treated samples that were cultured at 18° C. (data notshown).

Treatment with heat-stress and TSA mainly induced the formation of TypeII (up to 77% versus 7% in the control cultures) and Type IV structures(up to 32% versus 0.5% in the control cultures). Type I classicalembryogenic structures were observed at a low frequency when 0.5 μM TSAwas added to the culture medium (up to 1% versus 0,5% in the controlcultures), but were much more abundant when a ten times lowerconcentration of TSA was used (see FIG. 4B).

With the exception of Type III structures, all of the sporophyticmulticellular structures observed in control and heat-stress plusTSA-treated cultures were still present and had increased in size after15 days of culture, and were still more abundant in TSA-treated cultures(FIG. 3F, G). Types II and IV cell clusters eventually stopped growingand died. A very small percentage of heart to cotyledon stage embryoswere observed in the control cultures (up to 0.3%). Fewer embryos wereproduced in heat-stressed cultures treated with 0.5 TSA (up to 0.05%),but were up to five times more abundant than in control cultures whenheat-stressed cultures treated with the lower concentration (0.05 μM) ofTSA (FIG. 4C). Both the control and heat-stress plus TSA-treatedcultures contained classical type I embryogenic structures and theirnumbers can easily account for all the embryos formed; however, wecannot rule out that other types of cell clusters, such as the Type IIcallus-like structures, also develop into histodifferentiated embryos.

We determined whether the heat-stress treatment used to induce haploidembryogenesis is required for the TSA cell proliferation phenotype.Microspore cultures incubated at temperatures lower than 33° C. dividesporophytically, with the proportion of dividing cells depending on theculture temperature and stage of male gametophyte development, butproduce fewer or no embryos compared to 33° C. cultures. An increasedpercentage of sporophytic divisions appeared when TSA was applied tomicrospore cultures growing at either 18 or 25° C. (FIG. 4D-H), as wellas a corresponding increase in embryo production at 25° C. Up to 0.2%embryo production was observed in TSA-treated cultures compared topractically no embryo production in the non TSA-treated controls (FIG.4F). Higher TSA concentrations were needed to induce cell proliferationand embryo production at these lower temperatures compared to culturesgrown at 33° C.

Whilst not wishing to be bound to any particular theory, the inventorsconsider that TSA and heat-stress mediate similar developmental changesin microspore culture.

Example 3—Sporophytic Cell Clusters are Embryogenic

The cell clusters that are formed in heat-stressed, TSA treated culturesresemble those found in control cultures that are only exposed to aheat-stress treatment. They include classical embryogenic structures, aswell as structures that have been classified as non-embryogenic based ontheir unorganized structure, early release from the exine, and the lackof a protoderm, which is known to considered a hallmark for commitmentto embryo development in culture. Semi-quantitative RT-PCR and GFPreporter lines were used to determine whether the different types ofsporophytic structures that develop in control and TSA-treated culturesare embryogenic.

The expression of four embryo-expressed transcription factors genes,BABY BOOM (BBM); LEAFY COTYLEDON1 (LEC1); LEC2 and FUSCA3 is known to bepositively correlated with the embryogenic potential of B. napusmicrospore cultures. Semi-quantitative RT-PCR analysis showed thatexpression of these four genes was enhanced when microspore cultureswere treated with TSA, regardless of the culture temperature (data notshown) suggesting that TSA treatment is sufficient to activate theembryo pathway in microspore culture.

B. napus GFP reporter lines were then developed for two Arabidopsisembryo-expressed genes, LEC1 (LEC1:LEC1-GFP) and GLYCINE-RICH PROTEIN(GRP, GRP:GFP-GUS), to identify the specific structures that contributeto the enhanced embryo gene expression observed in TSA-treated cultures.The early embryo expression of both GFP reporters was confirmed in B.napus zygotic embryos, where LEC1 expression was detected as early asthe 2-cell stage and GRP expression from the zygote stage onward (datanot shown). Neither gene was expressed during the uni-, bi- ortrinucleate stages of male gametophyte development, either in theanther, or in microspore cultures grown at 18° C. to promote pollendevelopment.

The predominately nuclear localisation of the LEC1-GFP fusion was usedto more precisely follow the developmental identity of the differentcell types found in microspore cultures within the first three days ofculture. FIG. 5 shows micrographs of embryo-expressed GFP reporters inB. napus microspore culture (g=generative-like nucleus; scale bar in(A−J)=10 μm, (K−R)=25 μm). Panels A-H show expression of LEC1:LEC1-GFPin two day-old control (A, C, E, G) and TSA-treated (B, D, F, H, I, J)cultures. Panels (A, B) show microspore-like structure; (C, D)binucleate pollen-like structure; (E, F) trinucleate pollen-likestructure; (G, H) sporophytically-divided structure; (I)sporophytically-divided binucleate pollen-like structure showing GFP inthe two vegetative-like nuclei, but not in the generative-like nucleus;(J) sorophytically-divided binucleate pollen-like structure showing GFPin both the two vegetative-like nuclei and the generative-like nucleus;(K-R) LEC1:LEC1-GFP and GRP:GFP-GUS expression in five to eight day-oldTSA-treated microspore cultures treated with TSA; (K and L) Type Iembryogenic structures at eight days; (M and N) Type II compactcallus-like structures at eight days; (0 and P) Type III extrudedsporophytic structures at five days; (Q and R) Type IV loose callus-likestructure at eight days. For each panel, the image on the left side ofeach panel shows the GFP fluorescence and the image on the right side,the fluorescence from DAPI staining.

In control (heat-stressed) microspore cultures, LEC1-GFP was expressedin microspore-like structures, and in cells that contained two large,diffusely stained nuclei, but not in bi- or trinucleate pollen-likestructures (FIG. 5A, C, E, G). After TSA treatment of heat-stressedmicrospores, LEC1-GFP was also observed in the same structures as in thecontrol cultures, but also in bi- and trinucleate pollen-like structures(FIG. 5B, D, F, H). In pollen-like structures, LEC1-GFP was expressed ineither the vegetative-like nucleus or in both the vegetative- andgenerative-like nuclei, but never in generative-like nuclei alone (FIGS.5I and J).

Both the LEC1 and GRP reporters were expressed in the classical embryo(Type I) structures in the same spatial pattern as in zygotic embryos(FIGS. 5K and L), as well as throughout the Type II and IV sporophyticstructures. Only LEC1 expression was detected in Type III structures.The same pattern of expression was observed after TSA-treatment in oldercultures (FIG. 5M-R), An overview of the LEC1 and GRP expressionpatterns in control and TSA-treated cultures (data not shown) suggeststhat TSA-treated and control microspore cultures show similardevelopmental changes. Surprisingly, microspores can be reprogrammed toembryo development following heat-stress/TSA treatment in the absence ofcell division. Simultaneous exposure to TSA and heat-stress givesstronger effect than heat-stress alone, in that the embryo program isalso activated in both the vegetative- and generative-like cells ofimmature gametophyte.

Example 4—TSA Induces Totipotency in Arabidopsis Immature MaleGametophytes

Multicellular structures that resemble the Type II and IV structuresseen in Brassica microspore culture are produced when stage 11Arabidopsis anthers are cultured at 25° C. with 0.5 μM TSA. FIG. 6 showsmicrographs of five day-old anther cultures (scale bar (A, B)=50 μm, (C,D)=10 μm). TSA induces embryogenic cell divisions in Arabidopsisimmature male gametophytes. Panels show: (A)=DAPI stained anther inTSA-treated culture showing, multicellular, sporophytic structuresderived from immature male gametophytes (arrow). The insert shows aDAPI-stained multicellular structure with four nuclei. (B)=clearedanther from a control culture showing lack of spoorphytic cellproliferation. (C)=expression of LEC1:LEC1-GFP and (D)=ENOD4L:GFP in aType II compact callus-like structure in TSA-treated anthers. The exinestill surrounds the sporophytic structures (marked by arrows).

Growth of donor plants at a low temperature and in vitro culture at ahigher temperature, as in B. napus was not necessary, nor did it improvethe production of sporophytic structures. The percentage of immaturemale gametophytes that divided sporophytically in cultured Col0 antherswas highly variable (0-5%), but was never observed in anthers culturedwithout TSA (FIG. 6B). Expression of the LEC1 and GRP marker lines inTSA treated cultures was examined, but only LEC1 expression was detected(FIG. 6C). However, a third embryo reporter ENOD4-LIKE:GFP (ENOD4L:GFP)was expressed in the ISA-induced multicellular structures (FIG. 6D). TSAtherefore also induces embryogenic growth in Arabidopsis immature malegametophytes.

Example 5—Behaviour of Hda and Rbr Mutants in Arabidopsis Anther Culture

Arabidopsis contains 18 HDAC genes (referred to as HDA1-18) grouped intothe Rpd3/Hda1, HD-tuin and sirtuin families. This experiment determinedwhether T-DNA insertions in Arabidopsis HDAC genes phenocopy TSA-treatedanthers. Lines with T-DNA insertions in Rpd3/HDA1 and HD-twin type HDAgenes were examined for ectopic divisions of the male gametophyte duringnormal anther development in situ, but did not show any changes in thepollen cell division pattern in these lines.

FIG. 7 (scale bar=10 μm) shows behaviour of hda and rbr mutants inArabidopsis anther culture. The panels are as follows: (A)=shows theefficiency of sporophytic cell division in immature male gametophytes ofcultured anthers from hda T-DNA insertion lines treated with 0.5 μM TSA.Statistical comparison (Student's T-test) was made between theTSA-treated 0010 anthers and the ISA-treated hda mutant anthers,*p<0.05; **p<0.01; (B, C)=multicellular sporophytic structures observedin cultured rbr-3/+ anthers, rbr-like multicellular structure with threevegetative-like cells and one generative-like cell (B) and Type IImulticellular structure with eight nuclei (C); (D)=relative proportionof the different types of cells observed in rbr3/+ anther culturestreated with 0.5 μM TSA or DMSO (control cultures). Samples wereanalysed five days after the start of culture. Statistically significantdifferences were observed between the response of TSA treated anduntreated rbr-3 anthers (*, p<0.05; Student's T-test) and TSA treatedrbr-3 and 0010 anthers (+, p<0.05; Student's T-test).

It is currently difficult to test for TSA-independent or TSAhypersensitive responses in the single hda insertion lines due to thelow and variable response of the culture system. Given theselimitations, none hda insertion lines showed sporophytic divisions incultured pollen in the absence of TSA; however, when the same antherswere cultured in the presence of TSA, the hda17 T-DNA insertion lineshowed a small, but significant increase in the percentage ofsporophytic cell divisions relative to the control (FIG. 7A). This datasuggests that the activity of at least one HDAC, HDA17, is required tosuppress ectopic cell divisions in Arabidopsis pollen.

Experiments were done to see whether RBR plays a role in TSA-mediatedcell totipotency. Homozygous rbr mutants are gametophytic lethal,therefore the experiments were performed on heterozygous rbr anthers(rbr-3/+), which contain 50% rbr pollen. The developing structures werescored as dead, gametophytic, rbr-like or TSA-like. The rbr phenotype ismost penetrant during the bicellular stage of pollen development and ischaracterized by structures with multiple vegetative cells, and to alesser extent, extra generative-like cells (FIG. 7B). The TSA phenotypediffers from that of rbr in that the TSA-like cells are larger, containmore vegetative-like cells, and have a stretched or broken exine (FIG.7C). If an RBR-HDAC interaction is required to prevent sporophytic celldivisions in culture, then culturing rbr mutant pollen without TSA couldinduce TSA-like divisions. Culture of rbr-3 anthers with TSA should nothave an additive effect on the percentage of sporophytic divisions,except when TSA inhibition of HDAC activity is incomplete. Ectopic cellproliferation of immature male gametophytes was observed when rbr-3/+anthers were cultured in the absence of TSA. The typical compactrbr-like structures with up to 6 nuclei that develop in planta wereobserved (FIG. 7D). Strikingly, rbr-3/+ anthers cultured in the absenceof TSA also produced a low percentage (0,5%) of enlarged andloosely-connected Type II multicellular structures (FIG. 7D), which wasnever observed in cultured control anthers from wild-type plants. Nodifferences were observed between TSA-treated wild-type and TSA-treatedrbr-3/+ anthers, other than the typical rbr-like divisions that areobserved in the rbr3 line; however, compared to untreated rbr-3/+anthers, TSA-treated rbr-3/+ anthers showed a decrease in the frequencyof rbr-like divisions.

These experiments with cultured rbr3/+ anthers show that a loss of RBRfunction is sufficient to induce the formation of embryogenic cellclusters in Arabidopsis anther culture in the absence of TSA. Thedecrease in the frequency of rbr-like divisions after TSA treatment mayreflect a requirement for HDAC activity in promoting the typicalrbr-type cell-cycle progression.

Example 6—TSA Promotes Histone Acetylation

An acetylated lysine antibody was used in combination with protein gelblotting to identify proteins whose acetylation status changes in 8 hourheat-stress plus TSA-treated B. napus microspore cultures compared toheat-stressed control cultures.

FIG. 8 shows that TSA enhances histone acetylation. Panels show:(A)=Western blot of total acetylated proteins in microspore culturestreated for eight hours with DMSO (control) or TSA-proteins in the rangeof 10-25 kDa are differentially acetylated after TSA treatment comparedto the control; (B)=Western blot of total and acetylated (Ac) histone H3and H4 in microspore cultures treated for eight hours with DMSO(control) or TSA. The percentages of sporophytic divisions in thedifferent cultures at day 5 are shown under each sample.

Increased protein acetylation was observed in small molecular weightproteins in the range of 10-25 kDa in the TSA treated cultures comparedto control cultures (see FIG. 8A). The acetylation status of histones H3and H4 was determined during microspore culture using acetylated histoneH3 (Ac-H3) and H4 (Ac-H4) antibodies. Microspore cultures were startedfrom buds containing mostly binucleate pollen and placed for eight hoursat either 18° C. or 33° C. with or without 0.5 μM ISA. As expected, TSAgreatly enhanced sporophytic divisions at 18° C. and 33° C. compared tothe untreated controls (FIG. 8B). Although this increase in celldivision had no clear effect on the total amount of histone H3 and H4detected in the control and TSA-treated cultures, the level of histoneH3 and H4 acetylation increased dramatically in the ISA-treated culturesrelative to control cultures, both at 18° C. and at 33° C. (FIG. 8B).

The main effect of decreased HDAC activity following TSA treatment inmicrospore culture appears to be increased acetylation of histones.

Example 7—Effect of HDAC Inhibitors on Sporophytic Cell Division inMicrospore Cultures of B. napus DH 12075

FIG. 9 shows data for sporophytic cell divisions from microspores andpollen in the presence of DMSO (control) or different concentrations ofHDAC inhibitors after 5 days of microspore culture, Seven differentpopulations of microspores/pollen were tested and are ranked from (leftto right) the developmentally youngest to the developmentally oldeststages.

As shown in FIG. 9, I=Type I, classical embryo-forming structures (blackbars); II=Type II, compact callus-like structures (dark grey bars)III=Type III, extruded sporophytic structures (medium grey bars),IV=Type IV, loose callus-like structures (light grey bars) and V=pollen(white bars, F). Dead microspores and pollen were not included. Controlis a DMSO treated sample. The corresponding structures (I-V) are shownon the side panel.

Example 8—Effect of HDAC Inhibitors on Embryo Yield in MicrosporeCultures of B. napus DH 12075

FIG. 10 shows the embryo yield from microspores and pollen formed in thepresence of DMSO (control) or different concentrations of HDACinhibitors after 15 days of microspore culture. Seven differentpopulations of microspores/pollen were tested and are ranked (left toright) from the developmentally youngest to the developmentally oldeststages.

Example 9—HDACi Improve Embryo Quality in Older Stages of Donor Pollen

FIG. 11 shows data for microspore cultures from four stages ofdevelopment treated with either DMSO (control) or HDACi and scored forthe morphological type of embryo that was formed, as well as the yieldof embryos. The four different cultures (1-4) are ranked from (left toright) the developmentally youngest to the developmentally oldeststages. FIG. 11A shows the schematic representation of the differenttypes of embryo found in the four different cultures and at eachconcentration. Each embryo represents a yield of 0.1%. Each of thedifferent types of embryo was formed almost exclusively at the indicatedstage/concentration. FIG. 11B shows the types of embryo are: normal (i,v), rough (ii, vi), compressed (iii), ball-shaped (iv), cup-shapedcotyledons (vii) and reduced cotyledons (viii). For any given stage, atan optimum concentration HDACi treatment improves both the yield and thequality of the embryos that are formed relative to the control.

Example 10—HDACi-Treated Embryos can be Readily Converted to Seedlings

FIG. 12A shows the different morphological types of embryos produced inculture, whether from control or HDACi-treated cultures were able toproduce roots when transferred to germination medium, with the exceptionof the ball-shaped type of embryo. All of the embryos that producedroots, except for the short embryo type, initiated root development fromthe root meristem, as evidenced by the development of roots within a fewdays of growth on germination medium. However, short embryos stillproduced roots, but much later, indicating that the roots were producedindirectly via a callus phase. Notably, root growth in HDACi treatedembryos was more vigorous than in the similar type of control embryos(compare first two panels).

In FIG. 12B, different types of embryos derived from differentpopulations of microspores/pollen treated with DMSO (control) ordifferent concentrations of HDACi were transferred to regenerationmedium and evaluated for their ability to produce shoots (measure ofconversion). Embryos derived from HDACi-treated cultures showed similaror improved (SAHA) germination compared to the control.

The invention claimed is:
 1. A method of producing a haploid plantembryo comprising culturing or growing haploid plant material in thepresence of a hydroxamic acid compound, wherein the hydroxamic acidcompound is chosen from trichostatin A, suberoylanilide hydroxamic acid,suberoyl bis-hydroxamic acid, scriptaid, oxamflatin, tubacin, APHACompound 8, and apicidin.
 2. The method of claim 1, wherein thehydroxamic compound has histone deacetylase inhibitor activity.
 3. Themethod of claim 1, wherein the hydroxamic acid compound has haploidembryogenesis activity.